Aminoderivatives of biotin and their conjugates with macrocyclic chelating agents

ABSTRACT

Formula (I) compounds are described where the groups are as defined here below, processes for their preparation, and their uses for the preparation of conjugates with radionuclides for use in human and animal therapy and diagnostics, particularly for the diagnosis and therapy of pathological conditions such as tumours.

TECHNICAL FIELD

[0001] The invention described herein relates to modified biotins usefulfor the preparation of conjugates with radionuclides for use in humanand animal diagnostics and therapy, particularly for the diagnosis andtreatment of pathological conditions such as tumours.

[0002] The invention described herein relates to the technical field ofthe preparation of medicaments.

[0003] The invention described herein furnishes compounds, methods fortheir preparation, methods for their use and compositions containingthem, which are suitable for industrial application in thepharmaceutical field.

[0004] The invention described herein furnishes compounds, compositionsand methods suitable for the delivery and release of substances usefulin diagnostic and therapeutic medicine and in the treatment ofpathological disorders of organs and tissues.

[0005] In particular, though not exclusively, the invention describedherein relates to the field of anticancer radiopharmaceuticals, meaningboth substances which are useful for diagnostic purposes and substanceswhich are useful for cancer prevention and therapy.

BACKGROUND OF THE INVENTION

[0006] Tumour therapy is mostly implemented through the use ofsubstances targeted at destroying cancer cells. This can be achievedwith cytotoxic substances, which have to penetrate into the tumour cellsin order to exert their full effect, or by means of treatment of thetumour cells with radiation of sufficient energy to kill the cells. Inboth cases there is the problem of delivering the substance in asselective a manner as possible to the target cells, so as to avoidpossible damage to the surrounding healthy cells. In the case ofradiopharmaceuticals, i.e. substances carrying radioactive portions, theproblem of selectively delivering the active part (that is, theradioactive portion) to the tumour target, avoiding as far as possiblediffusion of the radionuclide in the body or interaction with healthycells surrounding the tumour, is perceived as being particularlyimportant.

[0007] For a discussion of all the issues involved and the solutionsproposed to date, the reader is referred to U.S. Pat. Nos. 5,283,342,5,608,060 and 5,955,605, assigned to Neorex, and based on a patentapplication filed on Jun. 9, 1992. These patents are specificallyincorporated herein for reference purposes.

[0008] In these documents, the problem, amongst others, of theresistance of the molecule carrying the radionuclide to the metabolicattacks of the body is discussed. Specifically, the case accorded mostattention is the molecule of biotin, which is one of the first choicesfor delivering the radionuclide to the tumour cells, thanks to itswell-known interaction with avidins. Biotin, as we know fromconsolidated practice, is bound to the radionuclide-chelating portion,e.g. a molecule of DOTA, via a linker. In fact, the Neorex patents posethe problem of the resistance of the complex consisting of the biotinmolecule, as connected to the radionuclide via the linker, tobiotinidases, enzymes that break the peptide bond present in thecomplex. This bond stems from the union of the chelating agent andbiotin.

[0009] Among its much desired characteristics, the molecule must beeliminated from the body rapidly and efficiently and must besufficiently small (m.w.<1000) to allow easy distribution into theextracellular fluid where it will bind with the tumour. In addition, itmust show proven stability in vivo with only minimal uptake bynon-tumour cells and rapid (renal) clearance and must not bemetabolised.

[0010] To these characteristics one should add the need for a certainamount of stability between the biotin part and the chelating portion ofthe molecule.

[0011] In fact, the chelating portion must not be released in vivo,freeing parts of the molecule, which are potentially dangerous for thebody. Experts in the field are clearly familiar with the problem of therelease of radionuclide by the chelating portion, including metal ionswhich are entirely foreign to the body, which may be endowed withradioactivity of various types and even high-energy radiation, which istherefore highly damaging.

SUMMARY OF THE INVENTION

[0012] It has now been found that the formula (I) compound, asrepresented here below, not only fulfills the requisites for such acompound in the therapy and diagnosis of tumours or other diseases whichcan be detected and treated with compounds of this type, but alsopresents the advantage of not undergoing metabolic reactions capable ofreleasing the complexing part of the molecule. In this way, the moleculewill be completely eliminated by the body in unaltered form, thusavoiding the problem of the possible release of the chelating part,containing the metal ions imprisoned within it.

[0013] One of the objects of the invention described herein is thereforea formula (I) compound:

[0014] where:

[0015] Q is a —(CH₂)_(n)— group, in which n is an integer from 4 to 12,in which case R′ does not exist, or Q is selected from the groupconsisting of —(CH₂)_(a)—CH(R′)—(CH₂)_(b)—, where a and b are,independently, integers from 0 to n, and R′ is defined here below, or Qis cyclohexyl, phenyl, in which case R′ is a substitute on thecyclohexyl or phenyl ring;

[0016] R is hydrogen or −Λ, where −Λ is a formula (II) macrocycle:

[0017] where the various Ys, which may be the same or different, areselected from the group consisting of hydrogen, straight or branchedC₁-C₄ alkyl, —(CH₂)_(m)—COOH, where m is an integer from 1 to 3; X ishydrogen, or the —CH₂-U group, where U is selected from methyl, ethyl,p-aminophenyl, or X is the —(CHW)_(o)-Z group, where o is an integerfrom 1 to 5, W is hydrogen, methyl or ethyl, Z is a heterocyclic groupwith 5 or 6 members containing one or more heteroatoms selected from O,N—R₁, R₁ being a hydrogen atom or a straight or branched C₁-C₄ alkylgroup, and S; or Z is selected from —NH₂, —NH—C(═NH)—NH₂, or —S—R₂ whereR₂ is a straight or branched C₁-C₄ alkyl group;

[0018] p is the integer 2 or 3

[0019] R′ is selected from the group consisting of hydrogen, straight orbranched C₁-C₄ alkyl, —(CH₂)_(q)-T, where T is selected from the groupconsisting of S—CH₃, —OH, or —COOH, and q is the integer 1 or 2;

[0020] R″ has the same meanings as R, with the following conditions:

[0021] if R is −Λ, R″ is hydrogen; if R is hydrogen, R″ is −Λ, or R andR″ are —(CH₂)_(r)-Λ (for R), where r is an integer from 4 to 12, and −Λ(for R″), respectively, Q being a —(CH₂)_(n)— group where n is aninteger from 4 to 12.

[0022] What is meant by a straight or branched C₁-C₄ alkyl group ismethyl, ethyl, propyl, isopropyl, n-butyl, sec-butyl, iso-butyl, orter-butyl.

[0023] What is meant by a heterocycle with 5 or 6 members is an aromaticor non-aromatic heterocycle having in the ring at least a heteroatomselected from O, N—R₁, or S, such as, for example, 2-, 3- or 4-pyridyl,or 2-, 4-, or 5-imidazolyl.

[0024] A first group of preferred compounds according to the inventionconsists in the formula (I) compounds where R is hydrogen, Q is—(CH₂)_(n)—, where n is an integer from 4 to 8, preferably 6, R″ is −Λ,Y is always —CH₂—COOH; X is hydrogen, and p is 2.

[0025] A further object of the invention described herein consists informula (I) compounds with radioisotopes for diagnostic and/ortherapeutic use. Examples of these isotopes are: Fe-52, Mn-52 m, Co-55,Cu-64, Ga-67, Ga-68, Tc-99m, In-111, I-123, I-125, I-131, P-32, Sc-47,Cu-67, Y-90, Pd-109, Ag-111, Pm-149, Re-186, Re-188, At-211, Bi-212,Bi-213, Rh-105, Sm-153, Lu-177, and Au-198.

[0026] A first group of preferred complexes according to the inventionare those where, in the formula (I) compounds, R is hydrogen, Q is—(CH₂)_(n)—, where n is an integer from 4 to 8, preferably 6, R″ is −Λ,Y is always —CH₂—COOH; X is hydrogen, p is 2 and the radioisotope isY-90.

[0027] Further objects of the invention described herein are processesfor the preparation of formula (I) compounds and their complexes withradiopharmaceuticals.

[0028] Further objects of the invention described herein arepharmaceutical and/or diagnostic compositions containing formula (I)compounds and their complexes as indicated above.

[0029] Other objects of the invention described herein are the use offormula (I) compounds and their complexes with radioisotopes asmedicaments or diagnostic tools, particularly for the preparation ofmedicaments which are useful in tumour therapy or diagnosis.

[0030] These and other objects relating to the invention describedherein will be illustrated in detail in the part that follows herebelow, also by means of experimental examples.

DETAILED DESCRIPTION OF THE INVENTION

[0031] The compound according to the invention described herein isprepared according to the following scheme, including the steps of:

[0032] a) formation of an amide bond between the carboxyl group ofbiotin and a primary amine group of H₂N-Q-NH₂ diamine, the other primaryamine group being suitably protected, for example, with a Boc group, ifnecessary.

[0033] b) deprotection of the primary amine group;

[0034] c) reduction of the amide group to an amine group;

[0035] d) conjugation with the desired formula (II) chelating agent −Λ.

[0036] Biotin is a commercial product. H₂N-Q-NH₂ diamines are availableon the market and can in any event be prepared using known methods.

[0037] The protection of the primary amine group is easily achievedusing known protective groups, such as, for example, Boc, and which inany event can be found in the sales catalogues and in the generalliterature.

[0038] Alternatively, the formula (I) compound according to theinvention can be prepared according to the following scheme, if R ishydrogen and R″ is a macrocyclic chelating agent −Λ:

[0039] a) formation of an amide bond between the carboxyl group ofbiotin and a primary amine group of H₂N-Q-NH₂ diamine, the other primaryamine group being suitably protected, for example, with a Boc group, ifnecessary;

[0040] b) deprotection of the primary amine group if the protectivegroup is of the alkyl urethane type, sensitive to treatment withBH₃.THF, such as, for example, a Boc group;

[0041] c) selective protection of said primary amine group with aprotective group selected from among those reported in the literature asbeing resistant to the subsequent reduction and detachable withoutdamaging the biotin ring (T. W. Greene, P. G. M. Wuts, “Protectivegroups in organic synthesis”, 3rd Ed., J. Wiley & Sons, Inc., New York,1999; Handbook of Reagents for Organic Synthesis, “Oxidizing andReducing Agents”, Edited by S. D. Burke and R. L. Danheiser, J. Wiley &Sons, Inc., New York, 1999);

[0042] d) reduction of the amide group to an amine group with BH₃.THF;

[0043] e) protection of the secondary amine group with protectionorthogonal to the preceding protective groups;

[0044] f) deprotection of the primary amine group;

[0045] g) conjugation with the desired chelating agent as defined above;

[0046] h) deprotection of the secondary amine group;

[0047] i) or, if R is a macrocyclic chelating agent −Λ and R″ ishydrogen, after step d):

[0048] j) conjugation with the desired chelating agent −Λ;

[0049] k) deprotection of the primary amine group.

[0050] The protection of the primary amine group in step a) has alreadybeen illustrated above. As regards the protection of the amine group instep c) and the protection of the secondary amine group, the averagetechnician is capable, on the basis of his or her knowledge of thefield, of selecting the appropriate protective group.

[0051] If R is —(CH₂)_(r)- Λ, and R″ is −Λ, the formula (I) compound canbe prepared according to the following scheme:

[0052] a) activation of the —COOH group of biotin according to the knownmethods of peptide synthesis (P. Lloyd-Williams, F. Albericio, E.Giralt, “Chemical Approaches to the Synthesis of Peptides and Proteins”,CRC Press, Boca Raton, New York, 1997);

[0053] b) conjugation of biotin activated with an amine with generalformula: BOcNH(CH₂)_(n)NH(CH₂)_(q)NHBoc, where n and q may vary,independently, from 4 to 12;

[0054] c) detachment of the protective group Boc;

[0055] d) reduction of amide, if desired, that can be performed asabove;

[0056] e) conjugation with the desired chelating agent −Λ.

[0057] A number of secondary amines illustrated in step b) can beobtained on the market; others can be prepared suitably modifyingconventional methods (for example see J. B. Hansen, , M. C. Nielsen, U.Ehrbar, O. Buchardt, Synthesis, 1982, 404).

[0058] The conjugation of the compound according to the invention withthe radioisotope to produce the complexes envisaged in the context ofthe invention described herein is carried out using the knowntraditional methods in the field, as described, for example, inPaganelli, Chinol et al. European Journal of Nuclear Medicine Vol. 26,No 4; April 1999; 348-357.

DESCRIPTION OF THE PREFERRED EMBODIMENT OF THE INVENTION

[0059] There now follows a detailed description of the preparation ofthe preferred formula (I) compound, that is to say the one in which R ishydrogen, Q is —(CH₂)_(n)—, where n is preferably 6, R″ is −Λ, Y isalways —CH₂—COOH; X is hydrogen, p is 2.

[0060] The process comprises:

[0061] a) formation of an amide bond between the carboxyl group ofbiotin and the primary amine group of hexamethylenediamine, suitablyprotected, for example with a Boc group, if necessary;

[0062] b) deprotection of the amine group of hexamethylenediamine;

[0063] c) reduction of the amide group to an amine group;

[0064] d) conjugation with the desired chelating agent.

[0065] Step a) in the process according to the invention describedherein consists in the formation of an amide bond between the biotincarboxyl group and the primary amine group of hexamethylenediamine-Boc.The biotin was treated with HATU to form an extremely active ester insitu that reacts with the amine group of hexamethylenediamine-Boc toform the relevant amide. This activation mechanism, which is used aboveall for peptide synthesis in the solid phase, requires a basic medium.To prevent the base from reacting with the active ester, tertiaryorganic bases such as di-isopropylethylamine (DIPEA) orN-methylmorpholin (NMM) are used. Protection of one of the two aminegroups of hexamethylenediamine with Boc (ter-butyloxycarbonyl) isnecessary to prevent the biotin binding to both ends of the diaminechain. The end product is isolated from the reaction medium afterevaporation of the solvent (DMF) and precipitation with water. Theproduct, recrystallised with propanol, was characterised by ¹H-NMR,elemental analysis and ESI-MS. The reaction yield is around 88%.

[0066] In step b), biotinyl-hexamethylenediamine-Boc is solubilised in amixture of AcOEt/HCl, approximately 3 M, to detach the Boc group. Afterremoving the solvent mixture the product was lyophilised to completelyeliminate HCl. The sample was purified by means of recrystallalisationwith an aqueous solution at basic pH and characterised by ¹H-NMR andTLC.

[0067] In step c), the reduction of the amide group was done withBH₃.THF. Since the reducing agent is extremely reactive, the processmust be carried out in anhydrous conditions. The starting product washeld under vacuum prior to the reaction and then solubilised inanhydrous THF (distilled with sodium and benzophenone). The reactionmixture was refluxed in a nitrogen atmosphere until complete reductionof the amide group (as monitored by ¹H-NMR spectra) had taken place.After evaporating the solvent under reduced pressure, the reactionmixture was treated with an aqueous solution of HCl. After lyophilisingthe acid solution, the product was purified by recrystallisation from anaqueous solution at basic pH and then by reverse-phase columnchromatography. Analysis of the product was done by analytical TLC whichrevealed its purity. The reaction yield is approximately 55%.

[0068] Step d) provides the conjugation reaction of the reducedbiotinyl-hexamethylenediamine with DOTA, performed with the specificreagents for the formation of amide bonds in an aqueous medium:1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC), andsulpho-NHS. DOTA was solubilised in water and adjusted to a pH betweenpKa₃ and pKa₄ values in order to activate mostly one of four carboxylicgroups. In this way, we can reduce the likelihood of obtaining sideproducts. To the basic solution were added sulpho-NHS and lastly EDC.After the formation of the active ester in situ, the reducedbiotinyl-hexamethylenediamine was added, checking that the pH of thesolution remained around 8.6. Purification of the crude product was doneby semipreparative HPLC (C₁₈; CH₃CN/H₂O/TFA 0.1%; CH₃CN from 5% to 25%in 20 min).

[0069] The objects of the invention described herein are pharmaceuticalor diagnostic compositions containing as their active ingredient atleast one formula (I) compound, also in the form of a complex with aradioisotope or, in the case of said formula (I) compound, inassociation with other active ingredients useful in the treatment of thediseases indicated in the invention described herein, e.g. otherproducts possessing anticancer activity; also in separate dosage formsor in forms suitable for combined therapy. The active ingredientaccording to the invention will be in the form of a mixture along withsuitable vehicles and/or excipients commonly used in pharmaceuticaltechnology, such as, for example, those described in “Remington'sPharmaceutical Sciences Handbook”, latest edition. The compositionsaccording to the invention shall contain a therapeutically effectiveamount of the active ingredient. The dosages will be determined by theexpert in the field, e.g. the clinician or primary care physician,according to the type of disease to be treated and the patientscondition, or concomitantly with the administration of other activeingredients.

[0070] Examples of pharmaceutical compositions are those that allowparenteral or loco-regional administration. Pharmaceutical compositionssuitable for the purpose are solutions, suspensions, or lyophilisedforms to be reconstituted at the time of use.

[0071] Forms suitable for the industrial application of the inventionare kits for cancer radiotherapy, as, for example, described in EuropeanPatent 0 496 074, in the paper by Paganelli, Chinol et al. published inthe European Journal of Nuclear Medicine Vol. 26, No 4; April 1999;348-357, in U.S. Pat. No. 5,968,405 and in the relevant literature.

[0072] A further object of the invention described herein is a kit forthe therapy or diagnosis of tumours by means of radioactivity.characterised in that at least one of the components of said kitcontains a formula (I) compound or one of its complexes with a suitableradioisotope.

[0073] The compounds according to the invention are useful for thepreparation of therapeutic and/or diagnostic agents for the treatmentand diagnosis of tumours.

[0074] For example, they can be used in tumour treatment methods withanticancer radiopharmaceuticals, such as, for example, those describedin European Patent 0 496 074, in the paper by Paganelli, Chinol et al.published in the European Journal of Nuclear Medicine Vol. 26, No 4;April 1999; 348-357, in U.S. Pat. No. 5,968,405 and in the relevantliterature.

[0075] The following example further illustrates the invention.

EXAMPLE

[0076] The NMR spectra were recorded in DMSO-d₆ solution.

[0077] To a solution of biotin (1 g, 4.1 mmol) and NMM (0.451 ml, 1 eq)in anhydrous DMF was added a solution of N-Boc-hexamethylene-diamine HCl(1.03 g, 1 eq) and NMM (0.451 ml, 1 eq) in anhydrous DMF. After a fewminutes a solution of HATU (1.56 g, 1 eq) in DMF was added. The reactionmixture was stirred overnight at room temperature and then evaporatedunder reduced pressure. The oil thus obtained was crystallised by addingwater and recrystallised with n-propanol obtaining 1.6 g (3.6 mmol; 88%yield) of compound 1.

[0078] The product was pure by TLC inspection (eluent:DCM/MeOH=5:1;detected by fluorescamine or Cl₂/o-tolidine).

[0079] Mp: 174-176° C.; ¹H-NMR, δ_(H): 1.1-1.65 [14H, CH(CH₂)₃ andNHCH₂(CH₂)₄], 1.34 (s, 9H, tBu), 2.05 (t, 2H, CH₂CO), 2.54 (d, 1H,HCHS), 2.74-3.15 (6H, 2×CH₂N, HCHS and CHS), 4.12 (m, 1H, CHCHNH), 4.28(m, 1H, CH₂CHNH), 6.36 and 6.42 (two s, 2×biotin NH), 6.74 (t, 1H, BocNH), 7.74 (t, 1H, amide NH); ESI-MS: m/e calcd. [M+H]⁺443.1, found443.1.

[0080] Elemental analysis: Calcd. for C₂₁H₃₈N₄O₄S. 0,5H₂O: C 55.85; H8.7; N 12.4. Found: C 56.2; H 8.8; N 12.4.

[0081] To a suspension of biotinyl-hexamethylenediamine-Boc (1) (1.6 g)in AcOEt was added 37% aqueous HCl until a solution of approximately 3 Min HCl was obtained. The solution was held under magnetic stirring for30 min and then evaporated under reduced pressure. The oily product wasthen lyophilised with water, adjusted to pH 12 with NaOH 2M, and cooledwith ice, whereupon the solution was lyophilised once again. The solidobtained (compound 2) was treated several times with MeOH to eliminatethe salts present and then purified by precipitation by adding ethylether to the methanol solution, obtaining 1.1 g (90% yield) of compound2. The compound was pure at TLC on silica gel (eluent:n-propanol/AcOH/H₂O, 1:1:1) as assessed with a solution of fluorescaminein acetone at 366 nm and with Cl₂/o-tolidine.

[0082] M.p.: 179-182° C.; ¹H-NMR, in accordance with compound 1, lackingtBu signal.

[0083] To a solution of 8.8 ml of BH₃ 1 M in THF, held under a nitrogenatmosphere at 0° C., was added amine 2 (1.5 g, 4.3 mmol) finely powderedand suspended in 15 ml of anhydrous THF. The mixture was held undermagnetic stirring at 0° C. for approximately 30 min and then refluxeduntil the reaction was complete (the progress of the reaction waschecked by ¹H-NMR on aliquots of the reaction mixture treated hot withHCl 3 M and evaporated under reduced pressure). At the end of thereaction HCl 3 M was added; the reaction mixture was then refluxed for 3hours and evaporated under reduced pressure. The crude reaction product(compound 3) was precipitated with water at a pH of approximately 12 andpurified by RP-CC (LiChroprep RP-8, 40-63 82 m; eluent:H₂O/CH₃CN/TFA-92:8:0.1) obtaining 1.3 g (2.3 mmol, 55% yield) ofcompound 3, which was pure at TLC inspection (same process used forcompound 2).

[0084]¹H-NMR, δ_(H): 1.30-1.58 [16H, CH(CH₂)₄ and NHCH₂(CH₂)₄], 2.53 (d,1H, HCHS), 2.82 (7H, HCHS and 3×CH₂N), 3.05 (m, 1H, CHS), 4.12 (m, 1H,CHCHNH), 4.28 (m, 1H, CH₂CHNH), 6.38 (br, 2×biotin NH), 8.03 (br, NH₃⁺), 8.9 (br, NH₂ ⁺). ESI-MS: m/e calcd. [M+H]⁺ 328.23; found 328.2

[0085] To a solution of DOTA.3H₂O (100 mg, 0.2 mmol) in water, adjustedto pH 9.2, was added a solution of sulpho-NHS (86.8 mg, 0.4 mmol) in 1ml of water. After a few minutes a solution of EDC (76.7 mg, 0.4 mmol)in 0.5 ml of water was added dropwise and cooled with ice. The reactionmixture was stirred for approximately 20 min, after which a solution ofamine 3 (111 mg, 0.2 mmol) dissolved in 1 ml of water at pH 8.6 wasadded dropwise. After approximately 3 hours the reaction solution waslyophilised and crude product 4 was purified by reverse phase HPLC (C₁₈,A: 0.1% TFA in CH₃CN; B: 0.1% TFA in water; from 10 to 15% of B in 20min; Rt: 12.6 min) obtaining 53 mg (20%) of product, which was pure atTLC (same process used for compound 2). The test with fluorescamine inacetone to ascertain the presence of a primary amine group yieldednegative results.

[0086]¹H NMR, δH: 1.3-1.6 [16H, CH(CH₂)₄ and NHCH₂(CH₂)₄], 2.60 (d, 1H,HCHS), 2.8-2.9 (7H, HCHS and 3×CH₂N), 3.04 (br s, 16H, 8×DOTA-ring CH₂),3.11 (m, 1H, CHS), 3.61 (br s, 8H, 4×DOTA CH₂CO), 4.15 (m, 1H, CHCHNH),4.33 (m, 1H, CH₂CHNH), 6.40 and 6.44 (two s, 2×biotin NH), 8.27 (br,amide NH), 8.54 (br, NH₂ ⁺). FAB-MS: [M+H]+ calcd. 715.9; found 715.6.;ESI-MS: [M+H]+ found 715,4.

[0087] Elemental analysis. Calcd. for C₃₂H₅₈N₈O₈S.4TFA.H₂O: C, 40.41; H,5.43; N, 9.42. Found: C, 40.48; H, 5.45; N, 9.09.

[0088] Labelling tests, avidin binding tests, and serum stability testswere carried out with the compound illustrated in the foregoing example.

[0089] Binding studies.

[0090] Avidin was reacted with ⁹⁰Y-DOTA-biotin at increasing amounts oflabelled biotin. The presence of other radioactive peaks besides that ofthe avidin-biotin complex was checked by FPLC using the isocraticconditions above described. The radiopeak corresponding to the⁹⁰Y-DOTA-biotin/avidin complex showed a retention time of 9 ml whereasthe peak of the unbound ⁹⁰Y-DOTA-biotin eluted at 15 min.

[0091] The results of the affinity studies between avidin and the ⁹⁰Ylabelled biotin derivative of Example 1 at the natural 1:4 molar ratioand in molar excess of avidin (1:2) are summarized in Table 1. TABLE 1Molar ratios avidin/biotin-DOTA Avi:Biot. Molar ratio 1:2 1:4⁹⁰Y-DOTA-Biotin (μg) 1 2 Avidin (μg) 51 51 Binding (%) 99.8 99.7

[0092] Beginning with molar excess of avidin, only one peak in theradiochromatogram, corresponding to the avidin-biotin complex, wasobserved. The same FPLC profile was obtained with a two fold increasedamount of ⁹⁰Y-DOTA-biotin demonstrating that the natural affinity of thesystem was maintained.

[0093] Affinity studies.

[0094] The displacement of ⁹⁰Y-DOTA-biotin from avidin by the action ofnatural biotin (vitamin-H), starting from a ratio of 1:4 avidin:biotinwas studied. The complete binding at 1:4 avidin:biotin molar ratio wasinitially checked by size exclusion FPLC using the above mentionedconditions. Aliquots from this solution were added with increasing molaramounts of vitamin-H and after 15 min. of incubation at room temperatureeach of them was analyzed by FPLC. The extent of displacement wascalculated by the reduction of avidin-biotin radiopeak compared to therising of the displaced ⁹⁰Y-DOTA-biotin radiopeak. The results aresummarized in Table 2 TABLE 2 Affinity studies with biotin. Avi:Vit. HMolar ratio 1:2 1:4 1:8 1:16 1:32 ⁹⁰Y-DOTA- 0.25 0.25 0.25 0.25 0.25Biotin (μg) Coupled Avidin 6.4 6.4 6.4 6.4 6.4 (μg) Added Biotin 0.0470.094 0.188 0.376 0.752 (μg) Displacement 0.2 0.0 0.8 0.0 0.0 (%)

[0095] The results <1% were considered beneath the experimental error.

[0096] It can be seen that even a large molar excess of vitamin-H couldnot displace the ⁹⁰Y-DOTA-biotin already bound to avidin.

[0097] Stability studies.

[0098] 50 μl of the labelling mixture, corresponding to 1.85 MBq of ⁹⁰Ywere diluted 20 fold with saline or human serum and incubated at 37° C.Solution were analyzed at different time points, up to 144 hours afterlabelling. To perform the analysis, an aliquot of the incubation mixturewas added to a molar excess of avidin. The ⁹⁰DOTA-biotin/avidin complexratio vs. free ⁹⁰Y, was determined by FPLC as above described.

[0099] The stability studies showed that in saline, the radiolabel wasstill completely associated with the avidin-biotin complex up to 144 h.

[0100] In serum, only one radiopeak was initially detected in thechromatograms of the samples incubated up 48 h, however, afterwards asteady dissociation of ⁹⁰Y from DOTA-biotin was observed reaching amaximum of 55% at 144 h.

[0101] Beginning in the sample incubated 72 h, a second peak at lowerretention time (8.2 min.) was observed in the radiochromatogramindicating that the ⁹⁰Y activity was associated with an high molecularweight species, presumably serum transferrin.

1. Formula (I) compound:

where: Q is a —CH₂)_(n)— group, where n is an integer from 4 to 12, inwhich case R′ does not exist, or Q is selected from the group consistingof —(CH₂)_(a)—CH(R′)—(CH₂)_(b)—, where a and b are, independently,integers from 0 to n, R′ is as defined here below, or Q is cyclohexyl,phenyl, in which case R′ is a substitute on the cyclohexyl or phenylring; R is hydrogen or −Λ, where −Λ is a formula (II) macrocycle:

where the various Ys, which may be the same or different, are selectedfrom the group consisting of hydrogen, straight or branched C₁-C₄ alkyl,—(CH₂)_(m)—COOH, where m is an integer from 1 to 3; X is hydrogen, orthe —CH₂-U group, where U is selected from methyl, ethyl, p-aminophenyl,or X is the —(CHW)_(o)-Z group, where o is an integer from 1 to 5, W ishydrogen, methyl or ethyl, Z is a heterocyclic group with 5 or 6 memberscontaining one or more heteroatoms selected from O, N—R₁, R₁ being ahydrogen atom or a straight or branched C₁-C₄ alkyl group, and S; or Zis selected from —NH₂, —NH—C(═NH)—NH₂, or —S—R₂ where R₂ is a straightor branched C₁-C₄ alkyl group; p is the integer 2 or 3 R′ is selectedfrom the group consisting of hydrogen, straight or branched C₁-C₄ alkyl,—(CH₂)_(q)-T, where T is selected from the group consisting of S—CH₃,—OH, or —COOH, and q is the integer 1 or 2; R″ has the same meanings asR, with the following conditions: if R is −Λ, R″ is hydrogen; if R ishydrogen, R″ is −Λ, or R and R″ are —(CH₂)_(r)-Λ (for R), where r is aninteger from 4 to 12, and −Λ (for R″), respectively, Q being a—(CH₂)_(n)— group where n is an integer from 4 to
 12. 2. Complex of aformula (I) compound with a radioisotope useful for therapeutic and/ordiagnostic purposes.
 3. Complex according to claim 2, where theradioisotope is selected from the group consisting of Fe-52, Mn-52m,Co-55, Cu-64, Ga-67, Ga-68, Tc-99m, In-111, I-123, I-125, I-131, P-32,Sc-47, Cu-67, Y-90, Pd-109, Ag-111, I-131, Pm-149, Re-186, Re-188,At-211, Bi-212, Bi-213, Rh-105, Sm-153, Lu-177, and Au-198.
 4. Complexaccording to claims 2 and 3, where, in the formula (I) compound, Q is—(CH₂)_(n)—, where n is an integer from 4 to 8, preferably 6, Y is—CH₂—COOH and the radioisotope is Y-90.
 5. Pharmaceutical and/ordiagnostic composition containing a 10 compound according to claim 1 or2, in a mixture with suitable vehicles and/or excipients.
 6. Use of thecompound according to claim 1 or 2 for the preparation of a drug usefulin the treatment of tumours.
 7. Pharmaceutical and/or diagnosticcomposition containing a complex according to one of claims 2, 3 or 4,in a mixture with suitable vehicles and/or excipients.
 8. Use of acomplex according to one of claims 2, 3 or 4 as an anticancerradiopharmaceutical.
 9. Kit for the radiotherapy or diagnosis oftumours, characterised in that at least one of the components of saidkit contains a compound according to claim 1 or claim 2.